A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level

Bacterial contamination accounts for more than half of food poisoning cases. Conventional methods such as colony-counting and general polymerase chain reaction are time-consuming, instrument-dependent, and sometimes not accurate. Herein, we developed a novel one-pot toolbox with precision and ultra sensitivity (OCTOPUS) platform for foodborne pathogen detection based on the mechanism in which Cas12a nontarget binding unleashes its collateral DNase activity. We demonstrated its application on two widespread foodborne bacteria, namely, E. coli O157:H7 and Streptococcus aureus, using specific crRNA targeting rfbE and nuc gene, respectively. For better sensitivity, recombinase polymerase amplification (RPA) was integrated without product purification. This one-pot detection, that is, RPA reagent, crRNA, and ssDNA-FQ reporter are all in one tube with the subsequent addition of Cas12a enzyme, was able to detect genomic DNA at the attomolar level. It omits an extra cap-opening process to avoid practical inconvenience and possible cross-sample contamination. Moreover, we demonstrated this platform for a real food matrix. A simple water boiling method for genome extraction together with one-pot assay achieved a limit of detection value of 1 CFU/mL in less than 50 min. This OCTOPUS technique integrates bacterial genome extraction, preamplification based on RPA, and Cas12a/crRNA cleavage assay.